Hydrogel thin film containing extracellular matrix components

ABSTRACT

The thin film of the invention comprises a hydrate of a vitrified gel containing an extracellular matrix components, which can be integrated with a retainer as required. A hydrogel thin film containing an extracellular matrix components such as thin-film collagen hydrogel thin film, which is useful for a cell culture substratum and for preventing organ adhesion, can be easily prepared, and is excellent in expediency.

FIELD OF THE INVENTION

[0001] The present invention relates to a hydrogel thin film containingan extracellular matrix components. More particularly, the presentinvention relates to a novel hydrogel thin film containing anextracellular matrix components, which is useful for a cell culturesubstratum and prevention of organ adhesion, has an appropriate elasticstrength, can be easily prepared and can be used simply.

PRIOR ART AND PROBLEMS

[0002] Cell culture has conventionally been carried out in variousmanners for various purposes including development of various medicaltechniques and various medical drugs.

[0003] A method using an extracellular matrix components such ascollagen is known as a method for cell culture. In this method, in thecase of collagen for example, various trial efforts have been made toensure a three-dimensionality as closest as possible to forms ofbiological tissues or functional expression by alleviating restrictionsimposed as a two-dimensional planar culture for ordinary cell culture.

[0004] Regarding the culturing methods using collagen or the likeattracting the general attention as to usage thereof, however, in thecase of collagen hydrogel for example, there is a problem of difficultyin handling the collagen hydrogel itself because of the softness. It isnot therefore easy to prepare a cell culture substratum, and a moresimple method for utilization has not as yet been established.

[0005] Under such circumstances, the present inventors have carried outstudies from various points of view regarding utilization of anextracellular matrix components such as collagen. The object of thesestudies was to improve the conventional culturing method, and to achievea method for using a new matrix substance, which would permit easierpreparation of a cell culture substratum, be simply applicable, providesatisfactory Performance as a culture substratum, and be applicable alsofor preventing organ adhesion.

SUMMARY OF THE INVENTION

[0006] As means to solve the foregoing problems, the present inventionprovides a hydrogel thin film containing an extracellular matrixcomponents, which is a thin film comprising a hydrate of a vitrifiedmatrix gel containing an extracellular matrix components (claim 1).

[0007] The present invention provides also embodiments wherein thehydrogel thin film contains a plurality of extracellular matrixcomponents (claim 2), wherein the extracellular matrix components iscollagen (claim 3), wherein the hydrogel thin film has a cell culturemedium components (claim 4), and wherein the thin film is integratedwith a retainer (claim 5). There are provided embodiments of theretainer, wherein the retainer is an annular or a mesh shape (claim 6),wherein the retainer comprises a biological absorbing substance(S)(claim 7), and wherein the retainer has a circular opening whichcomprises a cylinder retaining and integrating the thin film (claim 8).

[0008] The present invention provides also a glass-like substance of adried gel containing an extracellular matrix components as a precursorof the foregoing hydrogel thin film containing an extracellular matrixcomponents.

[0009] The present invention further provides, regarding the foregoinghydrogel thin film containing an extracellular matrix components, amethod comprising the steps of preparing the gel from a solutioncontaining the extracellular matrix components, drying the resultant gelfor vitrification thereof, and hydrating the vitrified gel (claim 10),and a method in which a hydrogel thin film containing an extracellularmatrix components is used as the cell culture substratum (claim 15).

[0010] The present invention further provides a method of culturingcells using the foregoing substratum.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows a phase-contrast microphotograph illustrating thesurface of a collagen hydrogel thin film as a reference;

[0012]FIG. 2 shows a perspective view illustrating a circular wireserving as a retainer;

[0013]FIG. 3 shows a photograph showing a collagen hydrogel thin filmsubstratum integrated with a circular wire retainer;

[0014]FIG. 4 shows a photograph showing a collagen hydrogel thin filmembedded gauze;

[0015]FIG. 5 shows a process diagram illustrating preparation of thesubstratum of the present invention using a cylinder as the retainer;

[0016]FIG. 6 shows a microphotograph showing formation of a colony forMEC; and

[0017]FIG. 7 shows a microphotograph showing the case of HDF.

[0018] In the drawings, the reference numerals represent the followingcomponents:.

[0019]1: Circular wire retainer

[0020]10: Cylindrical appliance

[0021]11: Circular wire retainer

[0022]12: Collagen hydrogel thin film

[0023]13: Cover

DETAILED DESCRIPTION OF THE INVENTION

[0024] As described above, the hydrogel thin film containing anextracellular matrix components of the present invention, as the thinfilm itself and as that integrated with the retainer, has an appropriateelastic strength and a thin film shape permitting easy handling, andenables achievement of a biochemical composition as a cell culturesubstratum, thus providing easy preparation for culturing and anexcellent expediency.

[0025] Collagen is a representative extracellular matrix components andits application is attracting the general attention. Applicableextracellular matrix components other than collagen include fibronectin,vitronectin, laminin, proteoglycan, glycosaminoglycan, and MATRIGEL(brand name), and may appropriately be used.

[0026] There is no particular restriction on the cell culture mediumcomponents, and optimum salt composition, concentration and pH areselected.

[0027] An annulus made of a wire or a metal line, or a mesh comprisinggauze or other woven stuff may be appropriately used as a retainer. Abiological absorbing substance may also be applicable. It suffices toselect a shape, a size and a material in response to the manner of use.The retainer may be a cylinder having a circular opening, or a containeras a precursor thereof.

[0028] Manufacture of a cell culture substratum comprises the steps offirst mixing an aqueous solution of a matrix such as collagen with acomposition having a medium or serum, placing any of various retainersas described above into this mixed solution, and incubating it at asuitable temperature for its gelation.

[0029] The resultant gel is vitrified by drying by air for example. Thisvitrification phenomenon, utilization of the thus vitrified gel afterfurther modification, and use of the modified gel after vitrification asa cell culture substratum are not known, but disclosed for the firsttime in the present invention.

[0030] More particularly, in the manufacturing method of the presentinvention, the vitrified gel containing an extracellular matrixcomponents such as collagen is hydrated. This provides a hydrogel thinfilm containing an extracellular matrix components having a satisfactoryelastic strength, which serves as a cell culture substratum and isuseful for preventing organ adhesion.

[0031] For vitrification, in the present invention, it is the generalpractice to slowly and completely dry the gel containing theextracellular matrix components having a terminal concentration in anaseptic manner (for example, through aseptic air drying), therebyvitrifying the gel. Hydration of the vitrified gel can easily beeffected by treating it by PBS, for example.

[0032] The construction and the functions of the present invention willnow be described below further in detail by means of examples involvingcollagen.

EXAMPLES Example 1 Preparation of Collagen Hydrogel Thin Film

[0033] 2 ml of quintuple-concentration Dulbecco's Modified Eagle Medium(GIBCO #31600-034), 0.1 ml of 10,000units/ml penicillin and 10,000 μg/mlstreptomycin (GIBCO #15140-031), 0.2 ml of 1M HEPES (GIBCO #15630-023),0.493 ml of 7.5% sodium bicarbonate solution (GIBCO #25080-011), 1.407ml of distilled water, and 1 ml of fetal bovine serum were put in asterilized conical tube (Falcon #2070) chilled on ice. having an innervolume of 50 ml, and mixed. Then 4.8 ml of 0.5% aqueous type-I collagensolution (CELLGEN I-AC or I-PC, made by Koken Company) was added intothe tube and uniformly mixed. After placing 2 ml of the mixed collagensolution having a final concentration of 0.24% in a culture dish made ofhydrophobic Polystyrene (φ35 mm; Falcon #1008), the solution was held ina humidified incubator at 37° C. in the presence of 5% CO_(2/)95% air in3 hours for its gelation. This collagen gel of the final concentrationof 0.24% was vitrified by completely air-drying slowly in an asepticmanner in the covered dish. The vitrified collagen gel was hydrated byadding 2 ml of PBS. The collagen gel thus hydrated was rinsed with 2 mlof PBS several times. The hydrated collagen gel was peeled off from thedish and collected by tracing the inner wall of the dish with asharp-end pincette along the periphery, in the form of a thin-filmcollagen hydrogel having a satisfactory elastic strength, as shown inthe phase-contrast microphotograph in FIG. 1.

Example 2 Preparation of Collagen Hydrogel Thin-Film with PeripheralWire Retainer

[0034] A circular retainer (1) with a knob as shown in FIG. 2 was madewith a stainless steel wire (size:#20; 0.9 mm), and 2 ml of the mixedcollagen solution having a final concentration of 0.24% prepared inExample 1 was placed, together with this wire retainer, into ahydrophobic polystyrene culture dish (φ35 mm; Falcon #1008). In the samemanner as in example 1, the colagen solution with the retainer wasvitrified ater its glation. The vitrified collagen gel was hydrated byadding 2 ml PBS. Further, the collagen gel thus hydrated was rinsedseveral times with 2 ml of PBS. The hydrated collagen gel could bepeeled off and collected from the dish by slowly lifting the stainlesssteel knob, in the form of a thin-film collagen hydrogel having asatisfactory elastic strength with a wire retainer surround it as shownin FIG. 3.

Example 3 Preparation of Gauze Embedding Type Collagen Hydrogel ThinFilm

[0035] A sterilized type-III gauze as prescribed in the JapanesePharmacopoeia (K-Pine made by, Kawamoto Hotai Zairyou Company) was cutin a circular shape with a knob in an aseptic manner, and immersed thecut gauze in a cell culture medium (Dulbecco's Modified Eagle Mediumcontaining 10% fetal bovine serum, 20 mM HEPES, 100 units/ml penicillinand 100 μg/ml streptomycin). The gauze was put, together with 5 ml ofthe mixed collagen solution having a final concentration of 0.24%prepared in Example 1, in a hydrophobic polystyrene culture dish (φ60mm; Falcon #1007). In the same manner as in example 1, the collagensolution with the gauze was vitrified after its gelation.

[0036] Furthermore, the vitrified collagen gel was hydrated by addingPBS in the same manner as in Example 1. The hydrated collagen gel couldbe peeled off-and collected from the dish by slowly lifting the knob ofthe gauze, in the form of a thin-film collagen hydrogel having asatisfactory elastic strength embedding the gauze as a whole as shown inFIG. A.

Example 4 Preparation Cylinder-Retained Collagen Hydrogel Thin-Film

[0037] A sterilized conical tube having an inner volume. of 50 ml(Falcon #2070) was cut to prepare a cylindrical appliance (10) capableof fixing a collagen hydrogel thin-film as a cell culture substratum asshown in FIG. 5 (a).

[0038] By the use of a collagen hydrogel thin film (12) with a wireperipheral retainer (11) prepared in Example 2, the cover (13) preparedfrom the conical tube was pressed as shown in FIG. 5 (b), and then thewire retainer (11) was removed. By doing so, it was possible to transferand fix easily the collagen hydrogel thin film onto an opening of theappliance (10). In this state, the resultant product could be used forcell culture. The gauze embedding type collagen hydrogel thin film shownin Example 3 could be fixed onto the appliance in the same manner asabove.

Example 5 Cell Culture on Collagen Hydrogel Thin Film

[0039] 10ml of the cell culture medium was placed in a hydrophilicpolystyrene culture dish (φ60 mm; Falcon #3002) , and the collagenhydrogel fixed onto the appliance in Example 4 was put into the dish. 2ml of the cell suspension (3×10⁴/ml) was seeded in the interior of theappliance and cultured in a humidified incubator at 37° C. in thepresence of 5% CO₂95% air. Human dermal fibroblasts (HDF) and humancholangio-adenocarcinoma cell line (MEC) were used as cells. Afterculturing the cells for five days, the cells were fixed with formalinand stained directly with hema-toxylineosin (HE). In the case of MEC,the cells formed several colonies on the collagen hydrogel thin film asshown in the photograph) in FIG. 6. In the case of HDF, some cellsseemed to invade-the collagen hydrogel thin film (photo in FIG. 7). Afrozen cross-section of the gel was therefore prepared and subjected toHE staining: while MEC showed no invasion into the gel, HDF revealed anapparent invasion into the gel.

[0040] It is needless to mention that the present invention is notlimited in any manner by the examples shown above. Various embodimentsare Possible as to cells capable of being cultured, composition ofculture medium, and culturing conditions as well as kinds of anextracellular matrix components such as a collagen hydrogel thin film,composition of substratum, thickness and elastic strength of the thinfilm.

[0041] According to the present invention, as described above in detail,there are available a culture substratum and an organ adhesionpreventive substance of a hydrogel thin film containing an extracellularmatrix components such as collagen, which can be easily prepared andprovides an excellent expediency.

What is claimed is:
 1. A hydrogel thin film containing an extracellularmatrix components, which is a thin film comprising a hydrate of avitrified matrix gel containing an extracellular matrix components.
 2. Ahydrogel thin film containing an extracellular matrix components asclaimed in claim 1, wherein said hydrogel thin film contains a pluralityof extracellular matrix components.
 3. A hydrogel thin film containingan extracellular matrix components as claimed in claim 1 or 2, whereinsaid an extracellular matrix components is collagen.
 4. A hydrogel thinfilm containing an extracellular matrix components as claimed in any oneof claims 1 to 3, wherein said hydrogel thin film has a cell culturemedium components
 5. A hydrogel thin film containing an extracellularmatrix components as claimed in any one of claims 1 to 4, wherein saidthin film is integrated with a retainer.
 6. A hydrogel thin filmcontaining an extracellular matrix components as claimed in claim 5,wherein said retainer is an annular or a mesh shape.
 7. A hydrogel thinfilm containing an extracellular matrix components as claimed in claim 5or 6, wherein said retainer comprises a biological absorbingsubstance(s).
 8. A hydrogel thin film containing an extracellular matrixcomponents as claimed in claim 5, wherein said retainer has a circularopening which comprises a cylinder retaining and integrating the thinfilm.
 9. A glass-like substance of a dried gel containing anextracellular matrix components, which comprises a dried gel containingan extracellular matrix component(s) as claimed in any one of claims 1to
 7. 10. A method of manufacturing a hydrogel thin film containing anextracellular matrix components, which comprises the steps of preparingthe gel from a solution containing the extracellular matrixcomponent(s), drying the resultant gel for vitrification thereof, andthen hydrating the vitrified gel.
 11. A method of manufacturing ahydrogel thin film containing an extracellular matrix components asclaimed in claim 10, wherein said hydrogel thin film contains aplurality of extracellular matrix components.
 12. A method ofmanufacturing a hydrogel thin film containing an extracellular matrixcomponents as claimed in claim 10 or 11, wherein said an extracellularmatrix components is collagen.
 13. A method of manufacturing a hydrogelthin film containing an extracellular matrix components as claimed inany one of claims 10 to 12, wherein said thin film has a cell culturemedium components.
 14. A method of manufacturing a hydrogel thin filmcontaining an extracellular matrix components as claimed in any one ofclaims 10 to 13, wherein the thin film is integrated with a retainer,which is prepared by gelation in the presence of said retainer andhydrating the vitrified gel after drying and vitrification.
 15. A methodof manufacturing a hydrogel thin film containing an extracellular matrixcomponents as claimed in claim 14, which is integrated with a cylinder,wherein said thin film retained integrally with said cyclic retainermanufactured by said method of claim 14 is fixed by the transfer ontothe cylinder having a circular opening having a diameter smaller thansaid cyclic retainer.
 16. A cell culture substratum which comprises thethin film as claimed in any one of claims 1 to
 8. 17. A cell culturingmethod using a hydrogel thin film containing an extracellular matrixcomponents, which comprises the step of seeding and culturing cells onthe substratum according to claim 16.